Odette Périn-Roussel

Tissue‐specific differences in adduct formation by hepatocarcinogenic and sarcomatogenic derivatives of 7H‐dibenzo[c,g]carbazole in mouse parenchymal and nonparenchymal liver cells

Parenchymal (PC) and nonparenchymal (NPC) liver cells have different tissue‐specific, procarcinogen activation enzymes. NPC appear to be protected against the mutagenic effects of lipotropic bulky adducts induced by carcinogens by a unknown mechanism. Most studies of activation have been conducted with whole liver. The purpose of this study was to differentiate adduct formation in mouse PC and in NPC, isolated after in vivo administration of 7H‐dibenzo[c,g]carbazole (DBC), the most efficient liver carcinogen in mice, which also has potent sarcomagenic and epitheliomagenic activities. The very sensitive 32P‐postlabeling method was used to detect adducts. Two tissue‐specific DBC derivatives, 6‐methoxy‐DBC (6MeODBC), which is exclusively sarcomagenic, and 5,9‐dimethyl‐DBC (DiMeDBC), which is exclusively hepatocarcinogenic, were analyzed in parallel. Both PC and NPC generated the ultimate metabolites of DBC, but NPC were substantially less efficient. Clear‐cut tissue‐specific differences in adduct formation were established: the sarcomagenic 6MeODBC gave rise only to NPC‐DNA adducts, and the hepatocarcinogenic DiMeDBC only to PC‐DNA adducts. The chromatograms of the adducts were compared with those of mouse embryonic cells in culture and mouse epidermal cells. The results are discussed in connection with animal experiments with DBC, 6MeODBC, and dimethylbenzanthracene and with published data on PC and NPC activating enzymes. Environ. Mol. Mutagen. 29:346–356, 1997.

DNA adduct formation in primary mouse embryo cells induced by 7H-dibenzo[c,g]carbazole and its organ-specific carcinogenic derivatives

The nuclease P1 modification of the 32P‐postlabeling technique was used to study the biological activity of 7H‐dibenzo[c,g]carbazole (DBC) and some of its derivatives, including N‐methyldibenzo[c,g]carbazole (N‐MeDBC), 5,9‐dimethyldibenzo[c,g]carbazole (5,9‐diMeDBC), 5,9, N‐trimethyldibenzo[c,g] carbazole (5, 9, N‐triMeDBC), 6‐methoxydibenzo[c, g]carbazole (6‐McODBC), N‐acetyldibenzo[c,g]carbazole (N‐AcDBC), N‐hydroxymethyldibenzo[c,g]carbazole (N‐HMeDBC) in primary mouse embryo cells. A very good correlation was found between carcinogenic specificity in vivo of these N‐heterocyclic aromatic hydrocarbons and their DNA‐adduction in vitro. Primary mouse embryo cells were able to metabolize and detect tissue‐specific sarcomagens N‐MeDBC and 6‐MeODBC as well as derivatives with both sarcomagenic and hepatocarcinogenic activity, DBC, N‐AcDBC, and N‐HMeDBC. The strong specific hepatocarcinogen 5,9‐diMeDBC in vivo, did not induce any DNA‐adducts in the embryo cells, which suggests that the enzymatic composition of the target tissue probobly is the determining factor in the organ specificity of this derivative. 5,9,N‐triMeDBC, derivative without any carcinogenic activity in vivo, did not induce any DNA‐adducts in primary mouse embryo cells. Pretreatment of cells with 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) apparently stimulated DNA‐adduct formation in the cells exposed to DBC, 6‐MeODBC, and N‐MeDBC. No or a very slight effect of TCDD on DNA‐adduct formation was found in cells exposed to N‐HMeDBC and N‐AcDBC. Preliminary results have shown that TCDD slightly induced cytochrome P4501A1 linked ethoxyresorufin O‐deethylase (EROD) activity in primary mouse embryo cells. These data suggest the role of cytochrome P4501A1 in the metabolism of DBC derivatives with sarcomagenic activity. Environ. Mal. Mutagen. 30:56–64, 1997

Tissue-specific activities of methylated dibenzo[c, g]carbazoles in mice : Carcinogenicity, DNA adduct formation, and CYP1A induction in liver and skin

The potent multitissue carcinogen 7H‐dibenzo[c,g]carbazole and nine methylated derivatives, synthesized on the basis of the positions in the parent compound that are involved in metabolism, were tested for their ability to induce sarcomas and hepatic tumors in XVIInc/Z mice. In addition, the capacity of these compounds to induce DNA adducts in skin and liver was investigated by 32P‐postlabeling analysis after their topical administration. Induction by these compounds of cytochromes P450 of the 1A family in liver and skin was investigated and correlated to their carcinogenic potential. A clear correlation was found between the tissue specificity of DNA binding and the capacity of each compound to induce skin or liver tumors. In contrast, no direct relationship was observed between the capacity of the compounds to induce cytochromes 1A1/1A2 and the tissue specificity of carcinogenesis or DNA binding in liver or skin. The results are discussed with respect to the positions of methyl groups in the 7H‐dibenzo[c,g]carbazole molecule. Environ. Mol. Mutagen. 35:139–149, 2000

Inhibition of 5,9-dimethyldibenzo[c, g]carbazole-DNA adduct formation in mouse liver by pretreatment with cytochrome P4501A inducers in vivo

Mice of the XVIInc/Z and DBA/2N strains, which are responsive and nonresponsive, respectively, to the aryl hydrocarbon (Ah) receptor, were treated with the hepatocarcinogen 5,9‐dimethyldibenzo[c,g]carbazole and their livers were examined by nuclease P1‐enhanced 32P‐postlabeling for the levels of DNA adducts formed. Pretreatment at the doses usually reported in the literature with the cytochrome P4501A (CYP1A) inducers 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD), β‐naphthoflavone (BNF), and isosafrole modulated DNA adduction. In XVIInc/Z mice, DNA adduction was totally inhibited by TCDD (a CYP1A1/1A2 inducer), BNF (a CYP1A1/1A2 inducer), and isosafrole (a CYP1A2 inducer). In DBA/2N mice, in which DNA adduction was also inhibited by TCDD, about 25% of the DNA adduct levels persisted after pretreatment with BNF (not a CYP1A1/1A2 inducer in this strain) or isosafrole (a CYP1A2 inducer in this strain). The increase (in all cases less than twofold) in the levels of the phase‐II drug‐metabolizing enzymes glutathione S‐transferase and uridine diphospho‐glucuronyltransferase after treatment with inducers cannot explain the total disappearance of DNA adducts. Assays of 5‐bromo‐2′‐deoxyuridine incorporation did not show any induction of DNA synthesis which could explain the decrease in adducts. These results suggest that in vivo 1) increases in CYP1A enzymes by inducers are not correlated with enhanced levels of certain DNA adducts; and 2) phase‐II drug metabolizing enzymes are not the main cellular protection pathway for detoxification. An additional mechanism, perhaps also induced by the Ah receptor but highly dependent on the dose of inducer, could be involved in parallel to multidrug resistance (mdr); further experiments are needed to identify this process used by the cell to enhance its protection against toxic or genotoxic effects. Environ. Mol. Mutagen. 32: 314–324, 1998

The metabolic activation of dibenzo[a,e]fluoranthene in vitro. Evidence that its bay-region and pseudo-bay-region diol-epoxides react preferentially with guanosine

Dibenzo[a,e]fluoranthene ( DBF ), a non- alternant carcinogenic polycyclic aromatic hydrocarbon (PAH), binds covalently to DNA. The main adducts were characterized as covalent additions of its bay-region and pseudo-bay-region diol-epoxides. The structure of these 2 adducts was analyzed by mass spectrometry using their persilyl derivatives. 3,4-Dihydroxy-1,2-epoxy-1,2,3,4-tetrahydro- DBF (3,4-diol-1,2-epoxy- DBF ) and 12,13-dihydroxy-10,11-epoxy-10,11,12,13-tetrahydro- DBF (12, 13-diol-10,11-epoxy- DBF ) obtained by synthesis were allowed to react in vitro with calf thymus DNA or with poly(G). The comigration of DNA and poly(G) adducts isolated after acid hydrolysis of DNA and poly(G) was in good agreement with mass spectroscopic results: both bay-region and pseudo-bay-region DBF diol-epoxides reacted with guanine residues.

Inversion of genetic predisposition to carcinogenesis in liver of two lines of mice selected for resistance (Car-R) or susceptibility (Car-S) to skin carcinogenesis.

Two lines of mice, one resistant (Car-R) and one susceptible (Car-S) to skin carcinogenesis, were produced by bi-directional selective breeding. To see whether the characteristics of susceptibility or resistance to tumorigenesis were also expressed in the liver and lung, the two lines were submitted comparatively to treatment with 5,9-dimethyl dibenzo[c,g]carbazole (DiMeDBC), a potent hepatocarcinogenic derivative of the ubiquitous heterocyclic carcinogenic pollutant, 7H-dibenzo[c,g]-carbazole (DBC). An inversion of genetic predisposition to carcinogenesis in liver was observed. Car-R animals displayed rapid tumorigenesis in 100% of cases while Car-S mice were remarkably less sensitive, showing a 4-fold lower mean tumor multiplicity and a 4-month longer latency time. In parallel adduct formation by DiMeDBC and DBC in liver DNA was analyzed by the 32P-postlabeling method, showing a remarkably higher level in Car-R mice than in Car-S animals. These data indicate that tissue-specific sensibility in carcinogenesis may involve gene expression at various levels.

Formation and persistence of DNA-protein cross-links induced in mouse fibroblasts by dibenzo [a,e] fluoranthene, and modulation by stimulators and inhibitors of polycyclic aromatic hydrocarbons (PAH) metabolism

The production by dibenzo[a,e]fluoranthene (DBF) of DNA-protein cross-links in cultured mouse fibroblasts is probably mediated by the activation of proximate metabolites of DBF and not by the DBF molecule itself. In order to test this hypothesis, several agents that enhance or reduce production of the DBF metabolite putatively involved in cross-linking were tested. Increasing NADPH concentrations in the medium enhanced cross-link production; 1,2-epoxy-3,3,3-trichloropropane (TCPO), an inhibitor of epoxide hydrolases, slightly reduced DNA-protein cross-link formation at high concentrations; norharman (NH), an inhibitor of certain steps in the metabolism of DBF, totally blocked cross-linking. The possible involvement of DBF-bisdihydrodiol, a bifunctional metabolite identified in vitro, is discussed. Postincubation in DBF-free medium did not induce a significant reduction in cross-links, indicating that repair did not take place.

Differences in metabolic activation of dibenzo[a,e]fluoranthene characterized by 32P-postlabeling in two mouse fibroblast models

The formation of DNA adducts was investigated in mouse fibroblasts from two different tissues--embryos and adult lung--after incubation with dibenzo[a,e]fluoranthene (DBF) or its major proximate metabolites. The nuclease P1 modification of the 32P-postlabeling method was adapted for detection of DBF-DNA adducts. Quantitative and qualitative differences were observed in the metabolic activation mediated by the two cell types. DBF-DNA adducts generated three major spots reproducibly, and more than ten spots of medium or weak importance. The highest level of DNA binding occurred via the DBF-bay region vicinal dihydrodiol epoxide but with significant differences in the quantitative distribution of adducts. Striking qualitative differences were observed when lung fibroblasts were incubated with the DBF-pseudo bay region dihydrodiol (DBF-12,13-DHD). The spots representing adducts induced in embryo fibroblasts by DBF-3OH-12,13-DHD, a further metabolite of DBF-12,13-DHD, were totally absent from chromatograms of lung cells. These results show that both embryo and lung fibroblasts can activate DBF but that different cytochrome P-450 forms and substrate affinities are involved. The finding that different activation systems may be present in subcategories of the same tissue, may provide a partial explanation for the wide variations in sensitivity to carcinogens among species, organs and tissues.